Aptamer is single-stranded DNA or RNA molecule. The aptamer is a small single-stranded oligonucleotide that can bind specifically to its target with high affinity. The aptamer can be used as a biosensor element capable of binding to a molecule in a detection/analysis system, and thus has been recognized as a substitute for antibody. Particularly, the aptamers can be used as molecules targeting various organic and inorganic materials, including toxins, unlike antibodies, and once an aptamer that binds specifically to a certain material is isolated, it can be consistently reproduced at low costs using automated oligomer synthesis methods. Since an aptamer-based biosensor of measuring a target protein using a fluorescence-labeled aptamer was first developed in 1996, various aptamer biosensors have been developed based on the advantages and structural properties of the aptamer (Yeon-Seok KIM & Man-Bock G U, NICE, 26(6):690, 2008).
To isolate such aptamers, a SELEX (Systematic Evolution of Ligands by EXponential enrichment) process has been used. However, in this SELEX process, aptamers for only single target molecule can be isolated, and an amplification/isolation process should be repeated 10 times or more until aptamers having high affinity are selected or a smaller number of nucleic acids remain, and also an affinity test is additionally required. Thus, the SELEX process has shortcomings in that it requires a large amount of time to develop a new aptamer and is complicated. Due to these shortcomings, there has been a demand for a new process for aptamer selection, which can simultaneously isolate aptamers for two or more target substances in a simpler and faster manner. In addition, the introduction of an automated process for aptamer selection has been demanded.
In recent years, several microfluidic techniques have been introduced for faster SELEX processes, and efforts have been made to shorten the time taken to isolate aptamers from several months/several weeks to several days (Hybarget, et al., Anal. Bioanal. Chem., 384:191, 2006; Windbichler, et al., Nat. Protoc., 1:637, 2006; Eulberg, et al., Nucleic Acids Research, 33:e45, 2005). However, such methods are not suitable as small-scale processes for multiplexed selection of aptamers. In connection with this, the present inventors developed a microfluidic device that employs a multiplex technique (see PCT/US2009/054097). However, this microfluidic device has shortcomings in that several aptamers cannot be simultaneously isolated, and that aptamers bound to target molecules can only be sequentially be isolated and also that means for isolating aptamers are limited. Thus, there has been a demand for the development of a new device and process, which use a new method suitable for shortening the process time and automating the overall process.
Accordingly, the present inventors have made extensive efforts to provide a new multiplex microfluidic device, which has improved efficiency and can be automated, and a method for high-throughput selection of aptamers using the same, and as a result, have found that a module assembly manufactured by constructing microfluidic device modules having a main microfluidic channel and a separate elution channel or tube and connecting the modules to each other can simultaneously isolate aptamers for several tens or hundreds of target substances in several tens or hundreds of chambers and can be automated by being connected to a high-throughput sequencing or high-throughput affinity test device, thereby completing the present invention.